Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation
Jin Xiang, Chang Chen, Rui Liu, Dongmei Gou, Lei Chang, Haijun Deng, Qingzhu Gao, Wanjun Zhang, Lin Tuo, Xuanming Pan, Li Liang, Jie Xia, Luyi Huang, Ke Yao, Bohong Wang, Zeping Hu, Ailong Huang, Kai Wang, Ni Tang
Jin Xiang, Chang Chen, Rui Liu, Dongmei Gou, Lei Chang, Haijun Deng, Qingzhu Gao, Wanjun Zhang, Lin Tuo, Xuanming Pan, Li Liang, Jie Xia, Luyi Huang, Ke Yao, Bohong Wang, Zeping Hu, Ailong Huang, Kai Wang, Ni Tang
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Research Article Metabolism Oncology

Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation

Abstract

Although cancer cells are frequently faced with a nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low-glucose conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation, promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation, counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.

Authors

Jin Xiang, Chang Chen, Rui Liu, Dongmei Gou, Lei Chang, Haijun Deng, Qingzhu Gao, Wanjun Zhang, Lin Tuo, Xuanming Pan, Li Liang, Jie Xia, Luyi Huang, Ke Yao, Bohong Wang, Zeping Hu, Ailong Huang, Kai Wang, Ni Tang

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Figure 7

PCK1 deficiency strengthens protein O-GlcNAcylation and correlates with human HCC growth.

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PCK1 deficiency strengthens protein O-GlcNAcylation and correlates with ...
(A) IHC staining of PCK1, O-GlcNAcylation, and Ki67 in clinical HCC samples. Scale bar: 100 μm. (B) Representative human HCC samples were indicated by immunoblots. (C) Relative PCK1 protein-expression levels were compared between nontumor tissues (NT) and tumors (T) from 40 patients with HCC (see also Supplemental Figure 11). Relative protein-expression levels were normalized to those in NT samples, followed by 2-tailed paired Student’s t test. (D) The correlation between HCC tumor sizes (n = 40) and PCK1 expression. Data are mean ± SD. P values were derived from Pearson’s correlation coefficient (r). (E) Relative O-GlcNAc levels of proteins in samples from 40 patients (see also Supplemental Figure 11). (F) Correlation analysis between PCK1 expression and O-GlcNAc levels (n = 40). Analysis of CHK2 O-GlcNAcylation in HCC tumors (n = 40, see also Supplemental Figure 11) by performing sWGA pull-down assays (G). CHK2 O-GlcNAcylation levels were quantified (H). (I) Correlation analysis between PCK1 and p-Rb expression in tumor tissues from 40 patients with HCC. (J) Molecular model for the role of PCK1 deficiency in regulating CHK2 O-GlcNAcylation and HCC growth upon low glucose. In a low-glucose microenvironment, PCK1 ablation promotes oxaloacetate accumulation and GFAT1 activation to increase UDP-GlcNAc synthesis through the hexosamine-biosynthesis pathway. Increased O-GlcNAc modification enhances Thr378 O-GlcNAcylation in CHK2, which leads to its dimerization and Rb phosphorylation, and HCC cell proliferation. Inhibitors AOA and DON suppress HCC growth, indicating a unique potential for targeting O-GlcNAc signaling in the treatment of HCC.